Mass Spectroscopy for Detecting Epigenetic Variations in Blood Cancer

Tamar Shahal, Chemistry, Tel Aviv University, Tel Aviv, Israel (tamarshahal.lab@gmail.com)
Yuval Ebenstein, Chemistry, Tel Aviv University, Tel Aviv, Israel

Epigenetic modifications include dynamic chemical alterations on specific DNA bases that do not involve changes in the DNA sequence. Specifically, 5-hydroxymethylcytosine (5-hmC) is a tissue-type specific epigenetic modification on the base cytosine. Reduction in 5-hmC levels has been recently associated with various types of cancer, such as leukemia, indicating its potential as a biomarker for early disease diagnosis. The challenges associated with detecting 5-hmC levels include: low ionization efficiency and interference from other nucleobases, in addition to its extremely low appearance frequency in gDNA. Liquid chromatography–tandem mass spectroscopy (LC-MS/MS) is considered to be the most reliable, quantitative technique for 5-hmC detection. However, the detection of 5-hmC levels in leukemia blood samples requires sensitivity which is challenging even for the LC-MS/MS technique. Here we introduce a simple enzymatic procedure for selective labeling of 5-hmC with a molecule of high ionization efficiency, resulting in eight folds increase in the LC-MS/MS response to 5-hmC. The limit of quantification (LOQ) of our technique is 0.001% 5-hmC per total nucleotides (below physiologically relevant levels), using only 500 ng DNA per measurement. We demonstrate that we can quantitatively differentiate between 5-hmC levels in healthy and leukemia blood samples (containing 0.006% and 0.003% 5-hmC/total nucleotides, respectively), using only 200 ng DNA per measurement. Thus, our method may open a new era of mass spectroscopy-based diagnostics for early detection of diseases associated with extremely reduced 5-hmC levels, such as leukemia. 

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Short Biography of Presenting Author

Dr. Tamar Shahal is a research associate in the lab of Dr. Yuval Ebenstein, Tel Aviv University. Recently she joined the founding team of the Sagol center for the epigenetics of metabolism and aging in Tel Aviv Sourasky Medical Center, as an expert in epigenetic profiling utilizing liquid chromatography- mass spectroscopy (LC-MS). She received her bachelor degree in Chemistry from Ben-Gurion University, Israel and her Ph.D. degree in Biotechnology from The University of Cambridge, UK where she worked on the development of an acoustic biosensor. Her postdoctoral research conducted in the Max Planck Institute for Intelligent Systems, Germany and in the Weizmann Institute of Science was focused on surface chemistries for studying cell behaviors. Her main focus today is the development of analytical methods for epigenetic mapping of gDNA utilizing LC-MS and various optical methods.